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Abstract:

The present invention relates to a polypeptide derived from a highly
conserved region (HCR) I-III of an extracellular region of a CD99 and
CD99 family such as CD99L2 and PBDX (or XG), which are a kind of
transmembrane protein, or a fused protein thereof. The polypeptide or the
fused protein thereof has an activating function of inhibiting the
extravasation of white blood cells, or inhibiting the growth and/or
metastasis of cancer cells. The present invention also provides a
polynucleotide coding the polypeptide, a vector including same, and a
transformant transformed by the vector. In addition, the present
invention provides a pharmaceutical composition including the polypeptide
or the fused protein thereof for preventing or treating inflammatory
diseases. Further, the present invention provides a pharmaceutical
composition including the polypeptide or the fused protein thereof
inhibiting the growth and/or metastasis of cancer cells, i.e., a
pharmaceutical composition for preventing or treating cancer.

Claims:

1. A polypeptide having an inhibitory activity against transmigration of
leukocytes or an inhibitory activity against growth and/or metastasis of
cancer cells, wherein the polypeptide is selected from the group
consisting of a polypeptide consisting of 3 to 96 amino acids derived
from the polypeptide of SEQ ID NO: 1, said polypeptide comprising the
peptide from position 28 to position 30 or from position 55 to position
57 of SEQ ID NO: 1, with the proviso that a polypeptide comprising the
peptide from position 94 to position 97 of SEQ ID NO: 1 is excluded; a
polypeptide consisting of 3 to 200 amino acids derived from the
polypeptide of SEQ ID NO: 2, said polypeptide comprising the peptide from
position 32 to position 34, from position 73 to position 75, from
position 121 to position 123, or from position 150 to position 152 of SEQ
ID NO: 2; and a polypeptide consisting of 3 to 130 amino acids derived
from the polypeptide of SEQ ID NO: 3, said polypeptide comprising the
peptide from position 27 to position 29 of SEQ ID NO: 3.

2. The polypeptide of claim 1, which is selected from the group
consisting of polypeptides as set forth in SEQ ID NOs: 4 to 14.

3. A fusion protein wherein a polyhistidine (poly-His) region is fused to
the polypeptide of claim 1.

4. The fusion protein of claim 3, wherein the poly-His region has the
amino acid sequence as set forth in SEQ ID NO: 15.

5. A fusion protein wherein a Fc region is fused to the polypeptide of
claim 1.

6. The fusion protein of claim 5, wherein the Fc region has the amino
acid sequence as set forth in SEQ ID NO: 16.

7. A polynucleotide encoding the polypeptide of claim 1.

8. The polynucleotide of claim 7, which is selected from the group
consisting of polynucleotides as set forth in SEQ ID NOs: 20 to 33.

9. A vector comprising a polynucleotide encoding the polypeptide of claim
1.

10. The vector of claim 9, wherein a cDNA encoding a poly-His region or a
Fc region is inserted.

11. A transformant obtained by transforming a host cell with the vector
of claim 9.

12. The transformant of claim 11, wherein the host cell is selected from
cells of microorganisms belonging to the genus Escherichia and the genus
Pichia.

13. A pharmaceutical composition for the prevention or treatment of
inflammatory diseases, comprising the polypeptide of claim 1, as an
active ingredient and a pharmaceutically acceptable carrier.

17. A pharmaceutical composition for the prevention or treatment of
inflammatory diseases comprising the fusion protein of claim 3, as an
active ingredient and a pharmaceutically acceptable carrier.

18. A pharmaceutical composition for inhibiting the growth and/or
metastasis of cancer cells, comprising the fusion protein of claim 3, as
an active ingredient and a pharmaceutically acceptable carrier.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a polypeptide or its fusion
protein having an inhibitory activity against transmigration of
leukocytes or an inhibitory activity against growth and/or metastasis of
cancer cells, the polypeptide being derived from highly conserved region
(HCR) I-III in the external domain of CD99 and its family, .i.e., CD99,
CD99L2, and PBDX(or XG). The present invention also relates to a
polynucleotide encoding the polypeptide, a vector including the
polynucleotide, and a transformant transformed with the vector. The
present invention also relates to a pharmaceutical composition for the
prevention or treatment of inflammatory diseases including the
polypeptide or its fusion protein. The present invention also relates to
a pharmaceutical composition for inhibiting the growth and/or metastasis
of cancer cells including the polypeptide or its fusion protein.

BACKGROUND ART

[0002] An inflammatory response is known as a protective response of
living organism for rehabilitating the structures and functions of
tissues damaged by infection, trauma, etc. Mobilization of leukocytes to
a focus of inflammation is critical for the rapid resolution of
infections and restoration of tissue damages resulting from a variety of
injuries. However, a misdirected or prolonged inflammatory response
causes damage to the body's tissues or diseases. For example,
inflammatory diseases are caused by bacterial or viral infection, e.g.,
cerebrospinal meningitis, enteritis, dermatitis, uveitis, encephalitis,
or adult respiratory distress syndrome, or non-infectious factors, e.g.,
trauma, autoimmune diseases, or organ transplantation rejection.
Inflammatory diseases are classified into acute and chronic inflammatory
diseases according to symptoms or pathological features. Acute
inflammation such as allergy or bacterial/viral infection is manifested
as local signs such as a change in bloodstream, blood vessel size, and
vascular permeability, and the recruitment of leukocytes. In contrast, a
main pathological feature of chronic inflammation such as rheumatoid
arthritis, artherosclerosis, chronic kidney infection, or hepatocirrhosis
is a continuous emigration of macrophages, lymphocytes, or plasma cells
into foci of inflammation due to recurrence of inflammatory factors,
thereby causing a long-lasting inflammatory response.

[0003] In order to induce an inflammatory response, the emigration of
leukocytes into inflammation foci is an essential event. Many cell
adhesion molecules are implicated in the emigration of leukocytes. That
is, the emigration of leukocytes includes a rolling stage in which
leukocytes are mobilized to the blood vessels of inflamed sites by
chemokine secreted from the inflamed sites and then rolled on surfaces of
vascular endothelial cells while reducing the velocity of cell movement;
an adhesion stage in which the leukocytes stops rolling and are firmly
adhered to the vascular endothelial cells; and a transmigration stage
wherein the leukocytes migrate through capillary vessels and basement
membranes. The final stage, i.e., the transmigration stage is also called
"diapedesis" or "transendothelial migration".

[0004] Cancer cells induced by carcinogens proliferate rapidly relative to
normal cells, thereby forming tumor masses, invading surrounding tissues,
and interfering with normal body functions. Cancer cells bring nutrients
and oxygen by inducing angiogenesis, and metastasis thereof is also
caused by angiogenesis. Although cancer cells grow infinitely at specific
sites, they can also leave the sites from which they originated, migrate
to and grow in new sites, whose process is called "metastasis".
Metastasis involve several key steps: conversion of cancer cells to
migratory mesenchymal cells, dissociation of the mesenchymal cells from
the original tumor sites, invasion into and spread through surrounding
connective tissues and capillary vessels, migration through blood
vessels, escape from the blood vessels, migration through connective
tissues, and proliferation in secondary sites.

[0006] The present inventors have disclosed that when CD99 is activated,
the function of β1 integrin is altered, thereby preventing the
adhesion of cancer cells onto extracellular matrices (ECMs). This
suggests that CD99 may be involved in metastasis of cancer cells (Suh J
S., 2001. Control of invasiveness of human breast carcinoma cell line
MCF-7 by CD99 molecule. Kangwon National University). In addition, the
present inventors have disclosed that a polypeptide derived from CD99,
i.e., a polypeptide comprising the peptide from position 94 to position
97 of CD99, can effectively activate CD99, thereby inhibiting the
transmigration of leukocytes or the growth and/or metastasis of cancer
cells (International Patent Publication No: WO 2007/037601).

[0008] The present inventors have found that a polypeptide or its fusion
protein having a certain amino acid sequence derived from highly
conserved regions (HCRs) of CD99 and its family, i.e., CD99, CD99L2, and
PBDX(or XG) can inhibit the transmigration of leukocytes, thereby
inhibiting inflammatory reaction; and also can inhibit angiogenesis and
the trans-endothelial migration of cancer cells, thereby inhibiting the
growth and/or metastasis of cancer cells.

[0009] Therefore, the present invention provides a polypeptide or its
fusion protein having an inhibitory activity against transmigration of
leukocytes or an inhibitory activity against growth and/or metastasis of
cancer cells, the polypeptide being derived from the HCRs of CD99,
CD99L2, and PBDX(or XG).

[0010] The present invention also provides a polynucleotide encoding the
polypeptide and a vector including the polynucleotide.

[0011] The present invention also provides a transformant obtained by
transforming a host cell with the vector.

[0012] The present invention also provides a pharmaceutical composition
for the prevention or treatment of inflammatory diseases, including the
polypeptide or its fusion protein as an active ingredient and a
pharmaceutically acceptable carrier.

[0013] The present invention also provides a pharmaceutical composition
for inhibiting the growth and/or metastasis of cancer cells (i.e., for
preventing or treating a cancer), including the polypeptide or its fusion
protein as an active ingredient and a pharmaceutically acceptable
carrier.

Technical Solution

[0014] In accordance with an aspect of the present invention, there is
provided a polypeptide having an inhibitory activity against
transmigration of leukocytes or an inhibitory activity against growth
and/or metastasis of cancer cells, wherein the polypeptide is selected
from the group consisting of a polypeptide consisting of 3 to 96 amino
acids derived from the polypeptide of SEQ ID NO: 1, said polypeptide
comprising the peptide from position 28 to position 30 or from position
55 to position 57 of SEQ ID NO: 1, with the proviso that a polypeptide
comprising the peptide from position 94 to position 97 of SEQ ID NO: 1 is
excluded; a polypeptide consisting of 3 to 200 amino acids derived from
the polypeptide of SEQ ID NO: 2, said polypeptide comprising the peptide
from position 32 to position 34, from position 73 to position 75, from
position 121 to position 123, or from position 150 to position 152 of SEQ
ID NO: 2; and a polypeptide consisting of 3 to 130 amino acids derived
from the polypeptide of SEQ ID NO: 3, said polypeptide comprising the
peptide from position 27 to position 29 of SEQ ID NO: 3.

[0015] In accordance with another aspect of the present invention, there
is provided a fusion protein of the polypeptide and a polyhistidine
(poly-His) region or a fusion protein of the polypeptide and a Fc region.

[0016] In accordance with still another aspect of the present invention,
there is provided a polynucleotide encoding the polypeptide.

[0017] In accordance with still another aspect of the present invention,
there is provided a vector comprising the polynucleotide encoding the
polypeptide.

[0018] In accordance with still another aspect of the present invention,
there is provided a transformant obtained by transforming a host cell
with the vector.

[0019] In accordance with yet another aspect of the present invention,
there is provided a pharmaceutical composition for the prevention or
treatment of inflammatory diseases, comprising the polypeptide or its
fusion protein as an active ingredient and a pharmaceutically acceptable
carrier.

[0020] In accordance with a further aspect of the present invention, there
is provided a pharmaceutical composition for inhibiting the growth and/or
metastasis of cancer cells, comprising the polypeptide or its fusion
protein as an active ingredient and a pharmaceutically acceptable
carrier.

Advantageous Effects

[0021] The polypeptide or its fusion protein according to the present
invention can inhibit the transmigration of leukocytes, thereby applying
to a pharmaceutical composition for inhibiting inflammation. And also,
the polypeptide or its fusion protein according to the present invention
can inhibit angiogenesis and trans-endothelial migration of cancer cells,
thereby inhibiting the growth and/or metastasis of cancer cells.
Therefore, the polypeptide or its fusion protein can be usefully applied
to a pharmaceutical composition for preventing or treating a cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows HCRs of the CD99 protein.

[0023] FIGS. 2 to 4 are the results obtained by evaluating the effects of
the polypeptides of the present invention on inactivation of β1
integrin.

[0024] FIGS. 5 to 7 are the results obtained by evaluating the effects of
the polypeptides of the present invention on adhesion between human
monocytes (U937) and human umbilical vein endothelial cells.

[0025] FIGS. 8 to 10 are the results of trans-endothelial migration assays
for human monocytes (U937), after treating with the polypeptides of the
present invention.

[0026] FIG. 11 shows the comparative values of ear weights, after
injecting the polypeptides of the present invention into acute contact
dermatitis-induced mice.

[0027] FIG. 12 is the results obtained by measuring the ear thickness
changes, after injecting the polypeptides of the present invention into
IgE-mediated immediate hypersensitivity reaction-induced mice.

[0028] FIG. 13 shows the results obtained by measuring the mean arthritis
scores, after administering the polypeptides of the present invention to
the mice having collagen-induced arthritis (CIA).

[0029] FIGS. 14 to 16 are the results obtained by evaluating the effects
of the polypeptides of the present invention on adhesion of human
umbilical vein endothelial cell (HUVEC) to fibronectin

[0030] FIGS. 17 to 19 are the results obtained by evaluating the effects
of the polypeptides of the present invention on angiogenesis of human
umbilical vein endothelial cell (HUVEC).

[0031] FIGS. 20 to 22 are the results of invasion assay for human breast
carcinoma cells (MCF-7), after treating with the polypeptides of the
present invention.

[0032] FIGS. 23 to 25 are the results of trans-endothelial migration
assays for human breast carcinoma cells (MCF-7), after treating with the
polypeptides of the present invention.

[0034] It is newly found by the present invention that the ligands of
various lengths derived from highly conserved regions (HCRs) of CD99 and
its family, .i.e., CD99 (SEQ ID NO: 1), CD99L2 (SEQ ID NO: 2), and
PBDX(or XG) (SEQ ID NO: 3) can inactivate β1 integrin through
binding to CD99 molecules. Furthermore, it is newly found by the present
invention that the polypeptide including a certain sequence derived from
HCRs of CD99 and its family can inhibit angiogenesis and transmigration
of cancer cells through inactivating the β1 integrin of
endothelial cells and cancer cells, thereby providing anticancer
activity.

[0035] Especially, it is also newly found that, according to sequence
analysis of the polypeptides, the sequence of Leu-Xaa-Asp is a minimum
unit for inactivating the β1 integrin. The Xaa may be any amino
acid, preferably Ser, Gly, Ala, or Glu, which are respectively the
peptides of SEQ ID NOs: 11 to 14. Therefore, it is found by the present
invention that the proteins derived from CD99, CD99L2, or PBDX(or XG)
comprising the sequence of Leu-Xaa-Asp can show anti-inflammatory
activity by blocking the transmigration of leukocytes through
inactivation of β1 integrin; and can show anticancer activity
by inhibiting angiogenesis of endothelial cells and transmigration of
cancer cells through inactivation of β1 integrin. Preferably,
the protein or its fusion protein may be applied to solid cancers such as
breast cancer, gastric cancer, colorectal cancer, colon cancer, rectal
cancer, and pancreatic cancer; or lymphoma.

[0036] The present invention provides a polypeptide having an inhibitory
activity against transmigration of leukocytes or an inhibitory activity
against growth and/or metastasis of cancer cells, wherein the polypeptide
is selected from the group consisting of a polypeptide consisting of 3 to
96 amino acids derived from the polypeptide of SEQ ID NO: 1, said
polypeptide comprising the peptide from position 28 to position 30 or
from position 55 to position 57 of SEQ ID NO: 1, with the proviso that a
polypeptide comprising the peptide from position 94 to position 97 of SEQ
ID NO: 1 is excluded; a polypeptide consisting of 3 to 200 amino acids
derived from the polypeptide of SEQ ID NO: 2, said polypeptide comprising
the peptide from position 32 to position 34, from position 73 to position
75, from position 121 to position 123, or from position 150 to position
152 of SEQ ID NO: 2; and a polypeptide consisting of 3 to 130 amino acids
derived from the polypeptide of SEQ ID NO: 3, said polypeptide comprising
the peptide from position 27 to position 29 of SEQ ID NO: 3. Preferably,
the polypeptide of the present invention may be selected from the group
consisting of polypeptides as set forth in SEQ ID NOs: 4 to 14.

[0037] The present invention also includes, within its scope, a fusion
protein of the polypeptide and a polyhistidine (poly-His) region or a
fusion protein of the polypeptide and a Fc region. The poly-His region,
which is a tag peptide, can be used for the separation and purification
of the polypeptide of the present invention by binding to a histidine
binding resin. In the fusion protein of the present invention, the
poly-His region may have the amino acid sequence as set forth in SEQ ID
NO: 15. The Fc region can be used for increasing stability in the blood
of the polypeptide. In the fusion protein of the present invention, the
Fc region may have the amino acid sequence as set forth in SEQ ID NO: 16.

[0038] The present invention also includes, within its scope, a
polynucleotide encoding the polypeptide. The polynucleotide can be
prepared from the nucleic acid sequences encoding CD99, CD99L2, or
PBDX(or XG), using a known method in the art. The polynucleotide may have
the nucleotide sequence as set forth in SEQ ID NOs: 20 to 33.

[0039] The present invention also includes, within its scope, a vector
comprising the polynucleotide encoding the polypeptide. Various known
cloning vectors, e.g., pPICZα A, B, or C (Invitrogen, U.S.A.), may
be used as a cloning vector. Preferably, a vector including DNA encoding
a poly-His region (e.g., SEQ ID NO: 34), for example, a pET28a(+) vector
(Novagen, U.S.A.) may be used as a cloning vector. And also, a vector
obtained by inserting DNA encoding a Fc region (e.g., cDNA consisting of
the nucleotide sequence as set forth in SEQ ID NO: 35) into a
conventional vector, e.g, a pET28a(+) vector (Novagen, U.S.A.) may be
used as a cloning vector. The vector of the present invention can be
constructed by inserting the polynucleotide encoding the polypeptide into
a cloning vector with an appropriate restriction enzyme site using a
method commonly known in the art. The vector of the present invention may
be directly used in a gene therapeutic composition for the purpose of
gene therapy or may be used in the production of transformants.

[0040] The present invention also includes, within its scope, a
transformant obtained by transforming a host cell with the vector. The
host cell is not particularly limited as long as the polypeptide can be
effectively expressed. Preferably, the host cell may be selected from
microorganisms belonging to the genus Escherichia (e.g., Escherichia
coli), the genus Pichia (e.g., X-33 Pichia; Invitrogen, U.S.A.), etc.

[0041] The present invention also provides a pharmaceutical composition
for the prevention or treatment of inflammatory diseases, comprising the
polypeptide or fusion protein as an active ingredient and a
pharmaceutically acceptable carrier.

[0042] The present invention also provides a pharmaceutical composition
for inhibiting the growth and/or metastasis of cancer cells, comprising
the polypeptide or its fusion protein as an active ingredient and a
pharmaceutically acceptable carrier.

[0043] The pharmaceutical compositions of the present invention may
include excipients such as lactose or corn starch, lubricants such as
magnesium stearate, currently available emulsifiers, suspending agents,
buffers, isotonic agents, etc. The pharmaceutical compositions of the
present invention can be administered orally or parenterally. Preferably,
the pharmaceutical compositions of the present invention can be
formulated into parenteral dosage forms. For intramuscular,
intraperitoneal, subcutaneous, or intravenous administration, a
sterilized solution of an active ingredient is generally prepared. In
this case, the sterilized solution may include a buffer to achieve a
desired pH value. With respect to formulations for intravenous
administration, an isotonic agent may be used to render the formulations
isotonic. The pharmaceutical compositions of the present invention can be
formulated into aqueous solutions including a pharmaceutically acceptable
carrier such as a saline of pH 7.4. The aqueous solutions can be
introduced into a patient's intramuscular blood stream by local bolus
injection.

[0044] The pharmaceutical composition of the present invention can be
administered to patients who suffer from various inflammatory diseases,
solid cancer (such as breast cancer, gastric cancer, colorectal cancer,
colon cancer, rectal cancer, pancreatic cancer) or lymphoma at a daily
dosage of about 1 to 2000 mg/kg. An adequate dosage is generally changed
according to age, body weight, and conditions of a patient.

[0045] Hereinafter, the present invention will be described more
specifically by the following working examples. However, the following
working examples are provided only for illustrations and thus the present
invention is not limited to or by them.

Example 1

Synthesis of Polypeptides

[0046] cDNA fragments of SEQ ID NOs: 20 and 21 encoding respective
polypeptides of SEQ ID NOs: 4 and 5 were inserted into pET28a(+)-Fc
vectors (prepared by inserting cDNAs of SEQ ID NO: 35, which encodes the
Fc regions of human immunoglobulin, into pET28a(+) vectors) to produce
pET28a-CD99L2EXT-Fc vectors and pET28a-PBDX(or XG)EXT-Fc vectors. That
is, the cDNA fragments of SEQ ID NOs: 20 and 21 were isolated by PCR,
digested with EcoRI, and inserted into the EcoRI sites of pET28a(+)-Fc
vectors with ligation enzymes to produce the pET28a-CD99L2EXT-Fc vectors
and pET28a-PBDX(or XG)EXT-Fc vectors.

[0047] Colonies obtained by transforming BL21(DE3) cells with the obtained
expression vectors were cultured in LB media for about 4 to 6 hours. When
the absorbance (A600) of the cultures reached 0.4-0.6, protein expression
was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) (1.4 mM)
for 7 to 9 hours. The cells were precipitated by centrifugation, washed
with phosphate buffered saline (PBS), and then re-precipitated to remove
impurities from the media. Fractions were analyzed by SDS-PAGE gel to
check protein expression.

[0048] For purification of expressed proteins, an 8M urea buffer (8M urea,
0.01M Tris-Cl, 0.1M NaH2PO4) was used. The pH of the urea
buffer was adjusted to 8.0, 6.3, 4.5, etc. according to a purification
step. The cells were lysed with a pH 8.0 urea buffer containing protease
inhibitors (1 mM PMSF, 10 μg/Ml leupeptin, 1 μg/Ml pepstatin, 1
μg/Ml aprotinin) and centrifuged at 13,000 rpm for 20 minutes at
4° C. The supernatants were mixed with histidine (His)-binding
resins (Ni-NTA His Bind Resins, Novagen, U.S.A.) in a 1 Ml Eppendorf
tube, and the mixtures were incubated at 4° C. for 16 hours to
induce the binding of histidine residues of the expressed proteins with
the His-binding resins. The reaction solutions were centrifuged, the
supernatants were discarded, and the pellets were washed with a pH 6.3
urea buffer. The protein was then dialyzed against PBS, and stored in
aliquots in a cold store.

[0051] Effects of the peptide fragments of SEQ ID NOs: 6 to 14 on
inactivation of β1 integrin expressed in human monocytes (U937)
were tested.

[0052] U937 cells (5×104) were added to each well and then
treated with the protein solutions including each peptide of SEQ ID NOs:
6 to 14 in PBS (5˜30 μg/Ml) prepared as in Example 2. After
incubation for 1 hour, the cells were washed with PBS three times and
then subject to lysis in 1% NP40 lysis buffer (1% Nonidet P40, 0.1M NaCl,
0.05M tris (pH 8.0), 5 mM EDTA) supplemented with 0.1 μM PMSP
(phenylmethylsulfonyl fluoride), 1 μg/Ml pepstatin A, 10 μg/Ml
leupeptin, 1 μg/Ml aprotinin, and 1 mM Na3VO4.

[0053] The cell lysates were subject to electrophoresis on 10%
polyacrylamide gel. For identifying an activated β1 integrin,
the electrophoresis was performed under non-reducing condition, i.e.,
without β-mercaptoethanol. The separated proteins were transferred
to a nitrocellulose membrane, and then treated with a blocking solution
(Tris-buffered saline (TBS) containing 0.05% Tween 20 and 3% bovine serum
albumin) at room temperature for about 1 hour. The proteins were
incubated for 2 hours, in a TBS buffer supplemented with
anti-β1 integrin monoclonal antibodies (Chemicon Co.; cat. No.
MAB2259Z) specific to the activated form of β1 integrin. After
washing with a TBS buffer containing 0.05% Tween 20, the proteins were
treated with horseradish peroxidase conjugated anti-mouse IgG (DiNonA
Co.; cat. No. 80019F) at room temperature for 1 hour. After washing five
times with a TBS buffer containing 0.05% Tween 20, the proteins were
visualized using an antibody detection kit (iNtRON Biotechnology, Inc).
For confirming the experiment on the same amount of cell lysate, actin
was also detected using anti-beta actin monoclonal antibodies
(Sigma-Aldrich Ltd.; cat No. A54441). The results are shown in FIGS. 2 to
4.

[0054] In FIGS. 2 to 4, QKKKLCF or LCF derived from the internal domain of
CD99 was used as a control peptide. Referring to FIGS. 2 to 4, in the
groups treated with the polypeptides of the present invention,
β1 integrin was inactivated in dose-dependent manner. However,
when treated with the polypeptide not including the amino acids of SEQ ID
NOs: 11 to 14, such a reduction was not observed (data not shown).

Experimental Example 2

Tests for Inhibitory Activity Against Adhesion Between Human Monocytes
(U937) and HUVECs

[0055] Effects of the peptide fragments of SEQ ID NOs: 6 to 14 on adhesion
between human monocytes (U937) and human umbilical vein endothelial cells
(HUVECs) were tested. HUVECs (5×104) were added to each well
of a 96-well culture plate. After incubation in 5% CO2 at 37°
C. for 24 hours, HUVECs were activated by treating with IL-1β for 4
hours, and then each well was washed with serum-free media. U937 cells
(1×105) were treated for 1 hour with the protein solutions
including each peptides of SEQ ID NOs: 6 to 14 in PBS (5˜30
μg/Ml) prepared as in Example 2. The resulting U937 cells were washed
with serum-free media three times and then added to each well containing
the HUVECs. After incubation for 1 hour at 37° C., the cells were
washed one time with PBS and then detached from the extracellular matrix
using trypsin-EDTA. The number of the U937 cells having small and
circular shape unlike HUVECs was determined under an inverted microscope,
using a hemacytometer. The results are shown in FIGS. 5 to 7. In FIGS. 5
to 7, QKKKLCF or LCF was used as a control peptide.

[0056] Referring to FIGS. 5 to 7, in the groups treated with the
polypeptides of the present invention, the number of monocytes adhered to
HUVECs was reduced by about 30˜60% relative to the control group.
And also, in case treated with the fusion proteins, i.e.,
pET28a-hCD99L2-Fc and pET28a-PBDX-Fc, similar results were obtained (data
not shown). However, in the group treated with the polypeptide not
including the amino acids of SEQ ID NOs: 11 to 14, such a reduction was
not observed. Thus, it is expected that the polypeptide including the
amino acids of SEQ ID NOs: 11 to 14 can inhibit trans-endothelial
migrations of the monocytes.

Experimental Example 3

Tests for Inhibitory Activity Against In Vitro Trans-Endothelial Migration
of Monocytes

[0057] HUVECs were cultured in the upper compartments of Boyden chambers.
The supernatants were removed, and human monocytes (U937), which had been
untreated or treated for 1 hour with the protein solutions including each
peptide of SEQ ID NOs: 6 to 14 in PBS (30 μg/Ml) prepared as in
Example 2, were seeded at 5×105 cells/chamber. At this time, a
culture including a supernatant obtained by centrifugation of a culture
obtained after culturing NIH/3T3 mouse fibroblasts in serum-free DMEM
containing 0.005% vitamin C and 0.1% bovine serum albumin for 16 hours
was placed in the lower compartments of the chambers to induce the
invasion of the monocytes. The chambers were incubated for 6 hours, and
the number of the cells migrated to the lower compartments was measured.
The test was repeated five times, and the results are shown in FIGS. 8 to
10. The control peptide is a peptide consisting of QKKKLCF or LCF.

[0058] Referring to FIGS. 8 to 10, trans-endothelial migrations of the
monocytes in the groups treated with the polypeptides of the present
invention were significantly reduced (about 25˜40% reduction) as
compared with that in the control group. Taking into consideration that
trans-endothelial migration is essential for migration of leukocytes into
inflammation sites through blood vessels, it is expected that the
polypeptides according to the present invention can effectively inhibit
the inflammatory reaction.

Experimental Example 4

Tests for Inhibitory Activity Against Acute Contact Dermatitis

[0059] Anti-inflammatory activities of the polypeptides according to the
present invention were investigated. 250 uM PMA (phorbol 12-myristate
13-acetate) was applied to one ear of BALB/c mice (about 6 weeks old) so
as to induce acute contact dermatitis. At the same time, the protein
solutions (100 μl) prepared by dissolving each peptide fragment of SEQ
ID NOs: 11 to 14 (100 μg) in PBS (100 μl) was injected through the
tail vain of the dermatitis-induced mice. A solution (100 μl) prepared
by dissolving the control peptide (i.e., QKKKLCF) (100 μg) in PBS (100
μl) was also injected in the same manner. After 6 hours, induction and
severeness of dermatitis was evaluated by measuring the ear weights. The
measurement of ear weights was performed by collecting the same size of
ear-samples from the three sites of each ear with a punch and then
weighing the obtained samples.

[0060] FIG. 11 is the graph obtained by comparing the ear weights of the
test group mice (treated with the polypeptides of the present invention)
with those of the control group mice (not treated with the peptide).
Referring to FIG. 11, in the groups treated with the polypeptides
according to the present invention, the ear weighs were reduced by about
15˜30% relative to the control group. Thus, it is expected that the
polypeptide of the present invention, including the peptides of SEQ ID
NOs: 11 to 14, can effectively inhibit the inflammatory reaction.

[0061] Anti-allergic activity of the polypeptide according to the present
invention was investigated. Balb/c mice (about 6 weeks old) were
sensitized by injecting IgE antibody (5 μg) through the tail vain
thereof. After 24 hours, the protein solution (100 μl) prepared by
dissolving the peptide fragment of SEQ ID NO: 13 (100 μg) in PBS (100
μl) was injected through the tail vain of the sensitized mice. A 0.15%
DNFB solution [2,4-dinitrofluorobenzene in aceton: olive oil (4:1)] as an
antigen was applied on each ear of the mice so as to induce IgE-mediated
immediate hypersensitivity. In case of the negative control mice, only
PBS (100 μl) was injected without treating a 0.15% DNFB solution. In
case of the positive control mice, PBS (100 μl) was injected and then
a 0.15% DNFB solution was treated. The changes in ear thickness were
measured with a digital caliper every hour for 12 hours. Every day from
the third day, the peptide fragment of SEQ ID NO: 13 (100 μg) was
intraperitoneally injected to the test group mice, while only PBS was
intraperitoneally injected to the control group mice. The changes in ear
thickness were measured for 15 days.

[0062] FIG. 12 is the graph obtained by comparing the ear thickness
changes of the test group mice and the positive control mice (increased
by treating a 0.15% DNFB solution) with those of the negative control.
Referring to FIG. 12, in the groups injected with the polypeptide
according to the present invention, the ear thickness increments were
remarkably decreased relative to the positive control group. Thus, it is
expected that the polypeptides according to the present invention,
including the peptide of SEQ ID NO: 13, can effectively inhibit an
IgE-mediated immediate hypersensitivity reaction.

[0063] Inhibitory activity of the peptide according to the present
invention against rheumatoid arthritis was investigated. A mixture (50
μl) having an equal volume (1:1) of CFA (Complete Freund's Adjuvant)
and Bovine type II collagen (2 mg/Ml) was injected subcutaneously in the
tail base of C57BL/6 mice (male, 4 weeks old). After 2 weeks, a mixture
having an equal volume (1:1) of Bovine type II collagen (2 mg/Ml) and
Incomplete Freund's Adjuvant was additionally injected into the sole of
the foot. When the mean arthritis score reached 9-12 by the induction of
CIA, mice were randomly divided into a test group and a control group.
The test group was orally administered with a solution of the peptide
fragment of SEQ ID NO: 13 (100 μg) in PBS (100 μl), while the
control group was orally administered with only PBS (100 μl). And
then, the mean arthritis scores were measured with naked eyes and
statistically analyzed for 21 days. The mean arthritis score was
assigned, based on the following criteria: 0=normal, 1=edema in less than
1 toe, 2=edema in more than 2 toes, 3=edema in the sole & edema in edema
in less than 1 toe, 4=edema in the sole of the foot & edema in more than
2 toes or edema in the sole of the foot and the ankle & edema in less
than 1 toe, 5=stiffness of tow.

[0064] FIG. 13 shows the results obtained by measuring the mean arthritis
scores of the control group (treated with only PBS) and the test group
(treated with the peptide according to the present invention). Referring
to FIG. 13, in the test groups orally administered with the peptide
according to the present invention, the arthritis was significantly
inhibited.

Experimental Example 7

Tests for Inhibitory Activity Against Adhesion of HUVECs to Extracellular
Matrix

[0065] Effects of the polypeptides of SEQ ID NOs: 4 to 14 on adhesion of
human umbilical vein endothelial cells (HUVECs) to fibronectin were
tested.

[0066] Each well of a 96-well culture plate was coated with fibronectin, a
component of extracellular matrix, and then dried under UV light. HUVECs
(5×104) were dispensed into each well, and then the protein
solutions including each peptide of SEQ ID NOs: 4 to 14 prepared as in
Example 2 were treated to each well, in the concentration of 3 μg/ml.
After incubation for 1 hour, the cells were washed three times with PBS,
detached using trypsin-EDTA, and then stained with a trypan-blue
solution. The number of the cells adhered to fibronectin was determined
using a hemacytometer. The results are shown in FIGS. 14 to 16. In FIGS.
14 to 16, QKKKLCF or LCF derived from the internal domain of CD99 was
used as a control peptide.

[0067] Referring to FIGS. 14 to 16, in the test groups treated with the
polypeptides of the present invention, the number of HUVECs adhered to
fibronectin was reduced by about 30˜60% relative to the control
group. And also, in case treated with the fusion proteins, i.e.,
CD99L2EXT-Fc and PBDX(or XG)-Fc, similar results were obtained (FIG. 16).
In this case, the control protein was a human IgG Fc, i.e., the protein
as set forth in SEQ ID NO: 16.

Experimental Example 8

Tests for Inhibitory Activity Against In Vitro Angiogenesis

[0068] Effects of the polypeptides of the present invention on
angiogenesis were evaluated.

[0069] Generally, interactions of basement membrane components of blood
vessels with vascular endothelial cells play an important role in
formation and maintenance of new blood vessels. When Matrigel, basement
membrane components, is treated to 24-well culture plate, plugs are
formed through polymerization reaction. HUVECs were seeded at a density
of 8×104 cells/well to each well of 24-well culture plates
coated with Matrigel. The protein solutions including each peptide of SEQ
ID NOs: 4 to 14 (30 μg/Ml) prepared as in Example 2 and bFGF (basic
fibroblast growth factor, 150 ng/Ml) were added to the wells. After
incubation for 24 hours, formation of new blood vessels was examined
using an inverted microscope (at 50× magnification), and the
results are shown in FIGS. 17 to 19. The control peptide and protein are
the same peptide and Fc as used in Experimental Example 1 and 7.

[0070] Referring to FIGS. 17 to 19, when HUVECs were treated with the
protein solution including the polypeptide of the present invention, tube
formation (i.e., angiogenesis) was significantly reduced. And also, in
case treated with the fusion proteins, i.e., CD99L2EXT-Fc and PBDX(or
XG)-Fc, similar results were obtained (FIG. 19).

Experimental Example 9

Tests for Inhibitory Activity Against Invasion of Cancer Cells

[0071] Each well of a transwell was coated with fibronectin, which is a
ligand of integrin. MCF-7 human breast cancer cells (5×105
cells) were loaded to the upper compartment of the transwell and then
incubated for 24 hours. When about 80% of the cells were grown up, each
well was treated with the protein solutions including each peptide of SEQ
ID NOs: 4 to 14 in PBS (30 μg/Ml) prepared as in Example 2. After
incubation in 5% CO2 at 37° C. for 1 hour, each well was
treated with 0.1% BSA. Invasion-inducing medium (the supernatant obtained
by incubating NIH/3T3 cells in the serum-free DMEM supplemented with
0.005% of vitamin C and 1% of BSA for 24 hours) was loaded into the lower
compartment. Cells migrated into the lower compartments of the transwell
were counted three times at 24-hour intervals, and then the results were
statistically analyzed. The control peptide and protein are the same
peptide and Fc as used in Experimental Example 1 and 7. The results are
shown in FIGS. 20 to 22.

[0072] Referring to FIGS. 20 to 22, in the groups treated with the
peptides of SEQ ID NOs: 4 to 14 according to the present invention, the
invasion rate of the human breast cancer cells was reduced by about 60%
relative to that of the control group treated with the control peptide.
Taking into consideration that cancer cells come out from blood vessels
and invade basement membranes or surrounding connective tissues and then
spread to secondary sites, it can be seen that polypeptides of the
present invention can effectively inhibit the metastasis of cancer cells.

Experimental Example 10

Tests for Inhibitory Activity Against In Vitro Trans-Endothelial Migration
of Cancer Cells

[0073] HUVECs were cultured in the upper compartments of Boyden chambers.
The supernatants were removed, and MCF-7 human breast cancer cells, which
had been untreated or treated for 1 hour with the protein solutions
including each peptide of SEQ ID NOs: 4 to 14 in PBS (30 μg/Ml)
prepared as in Example 2, were seeded at 5×105 cells/chamber.
At this time, the invasion-inducing medium was loaded into the lower
compartments of the chambers to induce the invasion of the breast cancer
cells. The chambers were incubated for 6 hours, and the number of the
cells migrated to the lower compartments was measured. The test was
repeated more than three times, and the results are shown in FIGS. 23 to
25. In FIGS. 23 to 25, the control peptide and protein are the same
peptide and Fc as used in Experimental Example 1 and 7.

[0074] Referring to FIGS. 23 to 25, trans-endothelial migrations of the
breast cancer cells in the groups treated with the polypeptides of the
present invention were reduced to about 60 to 80% of that in the control
group. Taking into consideration that trans-endothelial migration is
essential for migration of cancer cells into organs through blood
vessels, it can be seen that polypeptides of the present invention can
effectively inhibit the metastasis of cancer cells.